Phosphorylation is the most common covalent modification involved in regulation of metabolism in eukaryotes. This grant supports our studies on the structural and functional properties of protein kinases, their mode of regulation, identification of substrates, and the functional consequences of phosphorylation on activity. The specific aims to be addressed include the following. I. We will continue studies on the mode of regulation of casein kinase (CK) II by examining phosphorylation of the enzyme in vivo in response to hormones and in vitro in response to various effector compounds. The alpha and beta subunits of CK II are expressed in E. coli and will continue to be characterized alone and as the reconstituted holoenzyme and the effects of site-specific mutations on activity will be examined. II. Sequencing of the DNA coding for casein kinase (CK) I will be completed and the sequence compared with those of other protein kinases. We will continue the physical, chemical, and functional characterization of CK I. Phosphorylation and modulation of activity in cultured cells will also be examined. III. We will examine the effects of autophosphorylation of protease activated kinase (PAK) I on activity, determine whether the protein kinase is modified by other protein kinases, examine potential effectors of protein kinase activity and continue characterization of the enzyme. We will also examine the mode of activation of PAK I in cultured cells. IV. We will examine the requirements for complex association of protein substrates with other proteins/peptides to promote phosphorylation of the substrate and alter activity, using calmodulin as a model system. V. The effects of site-specific and multiple phosphorylation of proteins of interest will continue to be examined and the functional role of single and multiple phosphorylation events on enzyme activity, nucleic acid binding, and on protein/protein interactions will be analyzed.